RNase L is a principal mediator of the innate antiviral response and is thus critically important for human health. Virus replication in higher vertebrates is restrained by interferons (IFN) that cause cells to transcribe genes encoding antiviral proteins, including the 2'-5'oligoadenylate synthetases (OAS). The viral pathogen associated molecular pattern (PAMP), double-stranded RNA (dsRNA), activates OAS to produce 5'- phosphorylated, 2', 5'-linked oligoadenylates (2-5A) whose function is to stimulate RNase L. The OAS-RNase L system thus constitutes a classical innate immune pathway that rapidly responds to PAMPs (here, dsRNA) to produce a broadly active antiviral response. In our prior studies performed under this protocol, we cloned RNase L, knocked it out in mice, established its antiviral and apoptotic activities in vivo, and determined that it induces transcriptional signaling pathways to antiviral genes. Our future GOALS are to probe fundamental events and biologic endpoints surrounding RNase L that impact on viral lifecycles. Our HYPOTHESIS is that RNase L inhibits viral replication by cleaving viral and cellular RNA, initiating a stress-response pathway and signaling expression of antiviral genes. Our Specific Aims are to: (1) optimize novel small molecular 2-5A mimics that activate RNase L and determine their effects on cell viability and viral growth;(2) determine how 2-5A activation of RNase L signals transcription of antiviral and immunoregulatory genes, including investigations into the involvement of dsRNA signaling factors and MAP kinases;and (3) determine the fundamental basis for the antiviral activity of RNase L in vivo in wild type and RNase L-null mice by measuring viral induction of type I IFNs, determining the impact of RNase L-induced IFNs on viral replication and the effects of 2-5A and 2-5A mimics on IFN induction and viral replication. The experiments described in this PROPOSAL will investigate how a major antiviral pathway is stimulated by small molecule activators of a ubiquitous and potent antiviral enzyme (RNase L). The knowledge to be gained may thus lead to strategies for controlling important human viral pathogens. Therefore, there are cogent and health-related justifications for these studies.